Project summary DNA polymerase ? (Pol?) is a unique protein in higher eukaryotes because it contains a N-terminal superfamily 2 (SF2) helicase domain (Pol?-helicase) and a C-terminal A-family polymerase domain (Pol?- polymerase). Pol? is essential for the DNA double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also called microhomology-mediated end-joining (MMEJ). Pol? also promotes the proliferation of cancer cells defective in homology-directed repair (HDR) and confers resistance to chemotherapy agents. The structural and molecular basis of Pol? activity, however, remains unclear. For example, the activities of Pol?- helicase are poorly understood, and the structural basis for how Pol?-polymerase uniquely acts on single- strand DNA (ssDNA) overhangs and promotes MMEJ remains to be elucidated. Furthermore, how Pol? influences CRISPR-Cas9 genome editing remains poorly understood. In preliminary studies, we have discovered novel functions for Pol?-helicase including ssDNA annealing and DNA unwinding, and have identified optimal substrates for Pol?-polymerase MMEJ which will enable crystallographic studies of MMEJ. Lastly, we have discovered an unexpected and essential role for Pol? in CRISPR-Cas9 genome editing. We will utilize a collaborative approach among three labs (Pomerantz, Chen and Sfeir) to elucidate the structural and molecular basis of Pol? activity in DNA repair and genome editing by developing the following aims: 1. To investigate annealing, unwinding and anti-recombinase activities of Pol?; 2. To elucidate the structural basis of Pol?-polymerase dependent MMEJ; 3. To investigate Pol? involvement in CRISPR-Cas9 genome editing using ssDNA templates. In summary, these studies will provide new and significant insight into the structure and function of Pol?, and characterize an unexpected role for Pol? in CRISPR-Cas9 genome editing.